Method of Making Optically Active Ester Derivatives and Their Acids From Racemic Esters

ABSTRACT

The present invention relates to process for the preparing of optically active ester derivatives and their acid derivatives which are used intensively as important chiral intermediates from racemic β-hydroxybutyl ester derivatives. In more detail, this invention relates to the process for preparing optically active β-hydroxybutyl ester derivatives and their acid derivatives by stereospecific hydrolysis of racemic β-hydroxybutyl ester derivatives using Upases or lipase-producing microorganisms in the aqueous phase or organic phase including aqueous solvent. The method of making optically active ester derivatives and their acid derivatives by hydrolysis of β-hydroxybutyl ester derivatives represented by the general formula 1 in scheme 1 is easier and more economical comparing to the conventional methods and the products have high optical purity. Also separation of ester derivatives from acid derivatives is easy after reaction. Thus this method is a useful process on the industrial scale.

TECHNICAL FIELD

The present invention relates to a process for the preparation ofoptically active β-hydroxybutyl ester derivatives and their acidderivatives. In more detail, this invention relates to the process forpreparing optically active α-hydroxybutyl ester derivatives and theiracid derivatives by the hydrolysis of racemic α-hydroxybutyl esterderivatives represented by the general formula 1 in scheme 1 usinglipases or lipase-producing microorganisms.

The above-mentioned optically active α-hydroxybutyl ester derivativesand their acid derivatives can be used intensively as important chiralintermediates. Also, α-hydroxybutyl ester derivatives and their acidderivatives produced by this invention have high optical purity and thismethod can be used in practical process because separation and recoveryof the products are easy. Therefore this invention can be used on theindustrial scale.

According to a report, ethyl (R)-3-hydroxybutyrate is an intermediatefor an anti-glaucoma drug (Chirality in industry Π. Chichester,UK:Wiley, 1997, 245-262) and (S)-3-hydroxybutyrate is used forsynthesizing pheromones (Tertahedron, 1989, 45:3233-3298) andcarbapenems (Journal of the Chemical Society. Perkin Transaction, 1999,1: 2489-2494).

And ethyl (R)-4-chloro-3-hydroxybutyrate is used for synthesizingL-carnitine (Journal of the American Chemical Society, 1983,105:5925-5926), (R)-4-amino-3-hydroxybutyric acid (GABOB) and(R)-hydroxy-2-pyrrolidone. Ethyl (S)-4-chloro-3-hydroxybutyrate is avaluable synthon for the production of hydroxyymethylglutaryl CoA(HMG-CoA) reductase inhibitor (Journal of Medicinal Chemistry, 1990,33:2952-2956).

BACKGROUND ART

There are several methods to prepare optically active β-hydroxybutylester derivatives. Ethyl (S)-3-hydroxybutyrate is synthesized byasymmetric hydrogenation of ethylacetoacetate using BINAP-coordinatedRu(II) complexes (Journal of the American Chemical Society, 1987,109:5856-5858). However, this method has disadvantages of high pressureduring the reaction and high cost of metal catalyst.

Another method is the reduction of 3-oxo-esters using microorganisms.Jayasinghe et al. (Tetrahedron Letters, 1993, 34:3949-3950) obtainedethyl (S)-3-hydroxybutyrate (58% yield, 94% e.e) by the reduction ofethyl acetoacetate using freeze-dried yeast in petroleum ether andMedson et al.(Tetrahedron:Asymmetry, 1997, 8:1049-1054) obtained ethyl(S)-3-hydroxybutyrate (yield 69%, 99% e.e) from ethyl acetoacetate bythe reduction using yeast in organic solvent. Chin-Joe etal.(Biotechnology and Bioengineering, 2000, 69:370-376) obtained ethyl(S)-3-hydroxybutyrate (99% e.e) at 85% conversion by the reduction ofethyl acetoacetate using Baker's yeast. However, these methods havedisadvantages of low yield and purification problem after reaction.

On the other hand, Sugai et al.(Agricultural and Biological Chemistry,1989, 53:2009-2010) obtained ethyl (S)-3-hydroxybutyrate (99.4% e.e) bytransesterification of racemic ethyl 3-hydroxybutyrate using vinylbutanoate as an acylating agent and porcine pancreatic lipase as acatalyst. Fishman et al.(Biotechnology and Bioengineering, 2001,74:256-263) obtained ethyl (S)-3-hydroxybutyrate (40% yield, 99.4% e.e)using CALB (Candida antartica) lipaseand vinyl acetate as an acyl donor.

In another case, ethyl (R)-3-hydroxybutyrate can be prepared by acidicalcoholysis of Poly-(R)-3-hydroxybutyrate accumulated by microorganisms(Enzyme and Microbial Technology, 2000, 27:33-36).

Optically active ethyl 4-chloro-3-hydroxybutyrate can be produced byreduction of ethyl 4-chloroacetoacetate. Matsuyama et al.(Japan KokaiTokkyo Koho, 06-209782, Aug. 2, 1994) obtained ethyl(S)-4-chloro-3-hydroxybutyrate (97% yield, 98% e.e) using Kluyveromyceslactis NRIC 1329. Kataoka et al.(Applied microbiology and Biotechnology,1999, 51:486-490) obtained ethyl (R)-4-chloro-3-hydroxybutyrate (94%yield, 92% e.e) using recombinant microorganism, which coexpress boththe alcohol reductase Igene from Sporobolomyces salmonicolor and theglucose dehydrogenase gene from Bacillus megaterium. Yamamoto etal.(Bioscience Biotechnology and Biochemistry, 2002, 66(2):481-483)produced ethyl (R)-4-chloro-3-hydroxybutyrate (95.2% conversion, 99%e.e) using recombinant microorganism expressing secondary alcoholdehydrogenase from Candida parapsilosis. However, these methods havedisadvantage of long reaction time.

On the other hand, Hoff et al.(Tetrahedron:Asymmetry, 1999,10:1401-1412) obtained ethyl (S)-4-chlro-3-hydroxybutyrate (24% yield,86% e.e) by transesterifying for 5 days using Rhizomucor miehei lipase(RML) in organic phase (benzene).

Suzuki et al.(Enzyme Microbiology and Technology, 1999, 24:13-20)produced ethyl (R)-4-chloro-3-hydroxybutyrate (99.8% e.e) usingdechlorinase-producing microorganism.

As previously stated, optically active β-hydroxybutyl ester derivativescan be prepared by the stereoselective reduction of keto esters or theenzymatic transesterification. However, these methods are not suitabledue to their disadvantages including low enantiomeric excess, low yieldor difficulties in the separation of products and reactants afterreaction. For solving these problems, there is hydrolysis ofα-hydroxybutyl ester derivatives. Santaniello et al.(Gazzetta ChemicaItaliana, 1989, 119:581-584) obtained ethyl(R)-4-chloro-3-hydroxybutyrate and (S)-their acid by hyrolysis of ethyl4-chloro-3-hydroxybutyrate using pig liver esterase. However, thismethod has disadvantages of low yield (23%) and low enantiomeric excess(16% e.e) and is not suitable for industrial use.

DISCLOSURE OF INVENTION Technical Problem

The process for preparing of β-hydroxybutyl ester derivatives and theiracid derivatives of high optical purity was developed from racemicβ-hydroxybutyl ester derivatives represented by the general formula 1 inscheme 1 by stereospecific hydrolysis using lipases or lipase-producingmicroorganisms.

This method is simple and ester derivatives and their acid derivativesof higher optical purity can be obtained comparing to the conventionalmethods.

Accordingly, the objective of this invention is to provide the method ofpreparing optically active esters and their acids from racemicβ-hydroxybutyl ester derivatives using enzymes or microorganisms.

For the above objectives, the present invention consists of the processfor preparing high optically active β-hydroxybutyl ester derivatives andtheir acids from racemic β-hydroxybutyl ester derivatives bystereospecific hydrolysis using lipases or lipase-producingmicroorganisms as biocatalysts in aqueous phase or organic phaseincluding aqueous solvent.

Technical Solution

This invention is explained in more detail as follows. As mentionedabove, this invention relates to the process for preparing opticallyactive β-hydroxybutyl ester derivatives and their acid derivatives bystereospecific hydrolysis of racemic α-hydroxybutyl ester derivativesusing lipases or lipase-producing microorganisms as biocatalysts inaqueous phase or organic phase including aqueous solvent.

In this invention, methyl 3-hydroxybutyrate, ethyl 3-hydroxybutyrate,butyl 3-hydroxybutyrate, ethyl 3-azido-3-hydroxybutyrate, ethyl4-chloro-3-hydroxybutyrate, ethyl 4-bromo-3-hydroxybutyrate and ethyl4-cyano-3-hydroxybutyrate are used as racemic β-hydroxybutyl esters, butthe reactant is not restricted to them. In the general formula 1 inscheme 1, X is H, CN, N₃, F, Cl, Br or I and R is C_(n)H2_(n+1) (n=1˜8).

Non-limiting examples of the commercially available lipases include PSlipase from Amano Inc., Candida rugosa lipase and Novozyme 435 andnon-limiting examples of the lipase-producing microorganism includeCandida rugosa and Rhodococcus butanica.

After reaction, optically active esters and their acids are separatedrespectively by solvent extraction method or column chromatography.

In this invention, racemic compounds were determined by gaschromatography (Donam Instrument Inc. Model 6200) equipped with HP-FFAP(Agilent, Inc., 30 mm×0.53 m) column. The oven temperature wasmaintained initially at 70° C. for 5 min and then raised at the rate of10° C./min to 220° C., and maintained for 10 minutes. Helium gas is usedas carrier at the rate of 2 ml/min, and compounds were detected usingFID detector. The typical retention time of the components in thisinvention were as follows:

racemic methyl 3-hydroxybutyrate—15.48 min

racemic ethyl 3-hydroxybutyrate—14.32 min

racemic butyl 3-hydroxybutyrate—17.16 min

racemic ethyl 4-azido-3-hydroxybutyrate—22.50 min

racemic ethyl 4-chloro-3-hydroxybutyrate—20.31 min

Racemic ethyl 4-bromo-3-hydroxybutyrate and racemic ethyl4-cyano-3-hydroxybutyrate were analyzed using the same method used inthe analysis of racemic methyl 3-hydroxybutyrate except that oventemperature was increased at 20° C./min. In this condition racemic ethyl4-bromo-3-hydroxybutyrate and racemic ethyl 4-cyano-3-hydroxybutyrateare detected at 11.7 min and 14.07 min respectively.

Optically active methyl 3-hydroxybutyrate, ethyl 3-hydroxybutyrate,butyl 3-hydroxybutyrate and ethyl 4-azido-3-hydroxybutyrate weredetermined by HPLC (Waters, Inc., Model 1525) equipped with chial columnOD-H (Daicel, 0.46 cm×25 cm) using hexane and isopropyl alcohol mixture(90:10) as mobile phase. The absorbance was 220 nm and flow rate was 0.7ml/min. The typical retention time of the components in this inventionwas as follows:

methyl (R)-3-hydroxybutyrate—10.28 min

methyl (S)-3-hydroxybutyrate—12.44 min

ethyl (R)-3-hydroxybutyrate—12.77 min

ethyl (S)-3-hydroxybutyrate—11.43 min

butyl (R)-3-hydroxybutyrate—9.4 min

butyl (S)-3-hydroxybutyrate—10.64 min

ethyl (R)-4-azido-3-hydroxybutyrate-8.7 min

ethyl (S)-4-azido-3-hydroxybutyrate-10.86 min

Optically active ethyl 4-cyano-3-hydroxybutyrate was determined by a gaschromatography (Donam Instrument Inc. Model 6200) equipped with chiralcolumn G-TA (Astec, 30 mm×0.32 m). The oven temperature was maintainedinitially at 100° C. for 5 min and then raised to 170° C. at the rate of10° C./min, and maintained for 20 minutes. Helium gas was used ascarrier gas and column head pressure was maintained at 10 psi, andcompounds were detected using FID detector. In this condition, thetypical retention time of ethyl (R)-4-cyano-3-hydroxybutyrate and ethyl(S)-4-cyano-3-hydroxybutyrate was 16.75 min and 16.53 min respectively.

Optically active ethyl 4-chloro-3-hydroxybutyrate was determined by aHPLC (Lab Alliance, Model 201) equipped with chial column OB-H (Daicel,0.46 cm×25 cm) using hexane and isopropyl alcohol mixture (95:5) asmobile phase. The flow rate was 0.7 ml/min and absorbance was 215 nm.The typical retention time of ethyl (R)-4-chloro-3-hydroxybutyrate andethyl (S)-4-chloro-3-hydroxybutyrate was 14.42 min and 15.38 min,respectively.

Optically active ethyl 4-bromo-3-hydroxybutyrate was determined by aHPLC (Lab Alliance, Model 201) equipped with chial column AD-H(Daicel,0.46 cm×25 cm) using hexane and isopropyl alcohol mixture (90:10) asmobile phase. The flow rate was 0.7 ml/min and absorbance was 220 nm. Inthis condition, the typical retention time of ethyl(R)-4-bromo-3-hydroxybutyrate and ethyl (S)-4-bromo-3-hydroxybutyratewas 12.23 min and 11.24 min, respectively.

And racemic compounds were confirmed by FT-NMR (Burker, Model DRX300 orJEOL, Model AR400) and the results are as follows:

ethyl 4-azido-3-hydroxybutyrate

¹H-NMR (CDCl₃, 300 MHz) δ (ppm)=1.28 (t, 3H), 2.53 (m, 2H), 3.28 (d,1H), 3.34 (m, 2H), 4.21 (q, 2H)

ethyl 4-chloro-3-hydroxybutyrate

¹H-NMR (CDCl₃, 400 MHz) δ (ppm)=1.28 (t, 3H), 2.62 (d, 2H), 3.53 (br,1H), 3.60 (d, 2H), 4.20 (q, 2H), 4.33 (m, 1H)

ethyl 4-bromo-3-hydroxybutyrate

¹H-NMR (CDCl₃, 400 MHz) δ (ppm)=1.28 (t, 3H), 2.7 (m, 2H), 3.48 (dd,1H), 3.51 (dd, 1H), 4.17 (q, 2H), 4.20 (m, 1H)

ethyl 4-cyano-3-hydroxybutyrate

¹H-NMR (CDCl₃, 300 MHz) δ (ppm)=1.26 (t, 3H), 2.5-2.7 (m, 4H), 4.18 (q,2H), 4.32 (m, 1H)

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as the limit of the present invention.

EXAMPLE 1

Racemic ethyl 3-hydroxybutyrate (1%, v/v) was added to the vialcontaining 5 ml potassium phosphate buffer (pH 8.0, 0.1 M) and Novozyme435 (4%, w/v). The reaction was carried out at 30° C. for 2 hours. Thereaction mixture was extracted with ethyl acetate and analyzed byabove-mentioned method. Ethyl (S)-3-hydroxybutyrate (97% e.e) wasobtained from organic solvent at 55% conversion. The aqueous solutionwas acidified with hydrochloric acid and extracted with organic solvent.After esterification, ethyl (R)-3-hydroxybutyrate with optical purity of80.4% e.e was obtained.

EXAMPLE 2-3

Instead of lipase used in Example 1, whole cells (20% (w/v)) were used.Lipase-producing microorganisms were isolated by cultivating in a mediumcontaining 1% tributyrin. Isolated strains assimilated tributyrin andmade clear zone. The strains were grown in LB medium or GYP mediumincluding glucose, and microorganisms were harvested by centrifuge andused as biocatalysts. The results are shown in Table 1. TABLE 1 Con- %e.e Reaction version for Con- Example Microorgnism time(hr) (%) esterfiguration 2 Candida rugosa 62 69.6 99 S KCCM 50521 3 Rhodococcus 3.563.9 99 S butanica ATCC 21197

EXAMPLE 4-5

Instead of ethyl 3-hydroxybutyrate used in Example 1, 1% methyl3-hydroxybutyrate and 5% butyl 3-hydroxybutyrate were used as reactants.The reaction was carried out with novozyme 435 lipase and the resultsare shown in Table 2. TABLE 2 Con- % e.e Reaction version for Con-Example Reactant time(hr) (%) ester figuration 4 methyl 3- 5 78.0 98.8 Shydroxybutyrate 5 butyl 3- 6 82.0 78.5 S hydroxybutyrate

EXAMPLE 6

Instead of ethyl 3-hydroxybutyrate used in Example 1, ethyl4-azido-3-hydroxybutyrate was used. The reaction was carried out for 1hour and ethyl (S)-4-azido-3-hydroxybutyrate (80.2% e.e) was obtained at83.5% conversion.

EXAMPLE 7-8

Instead of ethyl 3-hydroxybutyrate used in Example 1, ethyl4-chloro-3-hydroxybutyrate was used as a reactant and lipases were usedas biocatalysts. The results are shown in Table 3. TABLE 3 Con- % e.eReaction version for Con- Example Lipase time(hr) (%) ester figuration 7Pseudomonas 22 71.0 99 S cepatia lipase 8 Candida rugosa 32 76.0 99 Rlipase

EXAMPLE 9

Instead of ethyl 3-hydroxybutyrate used in Example 1, ethyl4-bromo-3-hydroxybutyrate (1%, w/v) was used as a reactant and novozyme435 lipase was used as a biocatalyst. After reaction for 1 hour 40minutes, ethyl (R)-4-bromo-3-hydroxybutyrate (99% e.e) was obtained at88.3% conversion.

EXAMPLE 10

Instead of ethyl 3-hydroxybutyrate used in Example 1, ethyl4-cyano-3-hydroxybutyrate (1%, w/v) was used as a reactant and novozyme435 lipase was used as a biocatalyst. After reaction for 3 hours, ethyl(R)-4-cyano-3-hydroxybutyrate (99% e.e) was obtained at 57.3%conversion.

EXAMPLE 11-13

Instead of ethyl 3-hydroxybutyrate used in Example 2, ethyl4-chloro-3-hydroxybutyrate was used as a reactant and microorganisms inTable 4 were used as biocatalysts. The results are shown in Table 4.TABLE 4 Con- % e.e Reaction version for Con- Example Microorganism time(hr) (%) ester figuration 11 Candida rugosa 32 76.1 99 S KCTC 7292 12Candida rugosa 47 77.3 99 S KCCM 50521 13 Rhodococcus 8 76.1 99 Rbutanica ATCC 21197

INDUSTRIAL APPLICABILITY

In accordance with Examples 1-13, optically active β-hydroxybutyl esterderivatives can be produced easily by hydrolysis of this invention. Withappropriate lipases or microorganisms, β-hydroxybutyl ester derivativesof high optical purity can be produced. Also, it is easy to separateoptically active esters from their acids after reaction. Therefore, thismethod is a useful process on the industrial scale.

1. A process for preparing optically active β-hydroxybutyl esterderivatives and their acids derivatives from racemic β-hydroxybutylester derivatives represented by the general formula 1 in scheme 1 bylipases or lipase-producing microorganisms as biocatalysts in theaqueous phase or organic phase including aqueous solvent.

In the scheme 1, R is C_(n)H2_(n+1), (n=1-8) and X is H, N, CN, F, Cl,Br, I A process for preparing optically active β-hydroxybutyl esterderivatives and their acids derivatives according to claim 1, whereinmicroorganisms were Candida rugosa KCTC 7292, Candida rugosa KCCM 50521and Rhodococcus butanica ATCC 21197